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目的:探讨骨髓SSEA-1+干细胞向卵母细胞分化的潜能。方法:采用免疫磁珠分选系统(MACS)分选骨髓中SSEA-1+干细胞,接种于小鼠成纤维细胞(mE Fs)饲养层上,在含1 000 U/ml白血病抑制因子(LIF)的DMEM/F12培养液中将SSEA-1+干细胞传代培养至第2代(P2),撤去饲养层,在含20 ng/ml骨形成蛋白4(BMP4)的培养液中进行定向诱导,并分阶段加入体积分数10%人卵泡液、0.005 IU/ml卵泡刺激素(FSH)和0.003 IU/ml黄体生成素(LH)。光学显微镜下观察细胞形态学变化,检测不同发育阶段生殖细胞相关特异性基因Dazl、Fragilis、Mvh、Nobox、Oct4、Stella、STRA8、Gdf9的表达。结果:骨髓SSEA-1+干细胞生殖细胞相关特异性基因表达水平均显著高于骨髓单个核细胞和SSEA-1-细胞(P<0.05)。小鼠骨髓SSEA-1+干细胞与m EFs共培养后呈圆形,碱性磷酸酶染色阳性,显示细胞处于未分化状态。BMP4诱导7 d SSEA-1+干细胞形成细胞集落,14 d可见类胚体(EB)样结构形成,21 d可观察到卵母细胞样结构,且随诱导时间的延长生殖细胞相关特异性基因尤其卵母细胞减数分裂基因Stra8和Gdf9表达水平显著升高。结论:骨髓SSEA-1+干细胞具备向类卵母细胞样细胞分化的潜能。
Objective: To investigate the potential of bone marrow SSEA-1 + stem cells to differentiate into oocytes. Methods: The bone marrow SSEA-1 + stem cells were sorted by MACS and inoculated on the feeder layer of mouse fibroblasts (mE Fs). After the medium containing 1000 U / ml leukemia inhibitory factor (LIF) SSCs-1 + stem cells were subcultured to passage 2 (P2) in DMEM / F12 medium. After removal of the feeder layer, SSEA-1 + stem cells were induced in a culture medium containing 20 ng / ml bone morphogenetic protein 4 (BMP4) A volume fraction of 10% human follicular fluid, 0.005 IU / ml follicle stimulating hormone (FSH), and 0.003 IU / ml luteinizing hormone (LH) were added during the phase. The morphological changes of the cells were observed under an optical microscope to detect the expression of germ cell-related genes Dazl, Fragilis, Mvh, Nobox, Oct4, Stella, STRA8 and Gdf9 at different developmental stages. Results: The expression levels of SSEA-1 + stem cell germ cell-related genes were significantly higher than those of bone marrow mononuclear cells and SSEA-1 cells (P <0.05). Mouse bone marrow SSEA-1 + stem cells co-cultured with m EFs were round, positive staining for alkaline phosphatase, indicating that the cells in an undifferentiated state. BMP4-induced SSEA-1 + stem cells formed colonies on day 7, and embryoid bodies (EB) -like structures were observed on day 14, and oocyte-like structures were observed on day 21 and germ cell-associated genes The oocyte meiosis gene Stra8 and Gdf9 expression levels were significantly increased. Conclusion: Bone marrow SSEA-1 + stem cells have the potential to differentiate into oocyte-like cells.